The present invention relates to thiazolidinone compounds that inhibit telomerase activity, to pharmaceutical compositions containing the compounds and to the use of the compounds and compositions, alone or in combination with other pharmaceutically active agents, in the treatment of telomerase-mediated conditions or diseases, such as cancer.
Telomerase catalyzes the synthesis of telomeres. Telomeres are characteristic tandem repeats (TTAGGG in mammals) found at the ends of most eukaryotic chromosomes, that may be 15-25 kilobases long in human germline cells. With each cell division, about 60-100 bases are lost from the ends of the chromosomes, and as the telomeres shorten, cells eventually reach crisis and apotosis is triggered. See Harley et al., (1991) Mutation Res. 256: 271-282). Telomerase acts to maintain the telomere length just above the crisis level, and are thus responsible for chromosome stability and are involved in the regulation of the cell cycle.
Telomerase is a ribonucleoprotein reverse transcriptase that contains its own RNA template for the synthesis of telomeric DNA. See Blackburn, 1992, Annu. Rev. Biochem., 61:113-129. Telomerase is present in stem and germline cells of normal tissues, and at much higher levels in over 85% of tumors (Kim et al., 1994, Science, 266:2011-2014). Thus, drugs targeted towards telomerase potentially will have a high selectivity for tumor over healthy tissues. Consequently, telomerase inhibition has been proposed as a new approach to cancer therapy.
The inhibition of telomerase activity by antisense strategies directed towards the telomerase RNA component, for example peptide nucleic acids (Norton et al., (1996) Nature Biotech. 14: 615-619) and phosphorothioate oligonucleotides has been reported. Since telomerase is a reverse transcriptase, the use of inhibitors of reverse transcriptases, such as AZT, and other nucleosides has also been reported. Telomerase inhibition by cisplatin, possibly due to crosslinking of the telomeric repeat sequences, is also known (Burger et al., (1997) Eur. J. Cancer 33: 638-644).
Thiazolidinediones comprise a group of structurally related antidiabetic compounds that increases the insulin sensitivity of target tissues (skeletal muscle, liver, adipose) in insulin resistant animals. In addition to these effects on hyperglycemia, thiazolidinediones also reduce lipid and insulin levels in animal models of NIDDM. Recently, the thiazolidinedione troglitazone was shown to have these same beneficial effects in human patients suffering from impaired glucose tolerance, a metabolic condition that precedes the development of NIDDM, as in patients suffering from NIDDM (Nolan et al., (1994) N. Eng. J. Med. 331, 1188-1193). While their mechanism of action remains unclear, it is known that the thiazolidinediones do not cause increases in insulin secretion or in the number or affinity of insulin receptor binding sites, suggesting that thiazolidinediones amplify post-receptor events in the insulin signaling (Colca, J. R., and Morton, D. R. (1990) in New Antidiabetic Drugs (C. J. Bailey and P. R. Flatt, eds.). Smith-Gordon, New York, 255-261; Chang et al. (1983) Diabetes 32, 839-845).
Thiazolidinediones have been found to be efficacious inducers of differentiation in cultured pre-adipocyte cell lines (Hiragun et al. (1988) J. Cell Physiol. 134, 124-130; Sparks et al. (1991) J. Cell. Physiol. 146, 101-109; Kleitzien et al. (1992) Mol. Pharmacol. 41, 393-398). Additionally, thiazolidinediones have been implicated in appetite regulation disorders (see WO 94/25026 A1), and in increase of bone marrow fat content. In addition, thiazolidinedione compounds have been suggested for use in the treatment of psoriasis (U.S. Pat. No. 5,824,694) and climacteric symptoms and mesenchymal tumors (U.S. Pat. No. 5,814,647).
Some of the compounds useful for practicing the method of the present invention, and methods of making some of these compounds are known. For example, some of these compounds are disclosed in WO 91/07107; WO 92/02520; WO 94/01433; WO 89/08651; JP Kokai 69383/92; EP 0 155845; EP 0 155848; EP 0 193256; EP 0 295828; and U.S. Pat. Nos. 4,287,200; 4,340,605; 4,376,777; 4,438,141; 4,444,779; 4,461,902; 4,486,594; 4,572,912; 4,582,839; 4,687,777; 4,703,052; 4,725,610; 4,738,972; 4,775,687; 4,791,125; 4,812,570; 4,873,255; 4,897,393; 4,897,405; 4,918,091; 4,948,900; 5,002,953; 5,023,085; 5,053,420; 5,061,717; 5,120,754; 5,132,317; 5,143,928; 5,194,443; 5,223,522; 5,232,925; 5,252,735; 5,260,445; 5,814,647; 5,824,694; and 5,874,454.
The identification of compounds that inhibit telomerase activity provides important benefits to efforts at treating human disease. Compounds that inhibit telomerase activity can be used to treat telomerase-mediated disorders, such as cancer, since cancer cells express telomerase activity and normal human somatic cells do not possess telomerase activity at biologically relevant levels (i.e., at levels sufficient to maintain telomere length over many cell divisions). Unfortunately, few such compounds, especially compounds with high potency or activity and compounds that are orally bioavailable, have been identified and characterized. Hence, there remains a need for compounds that act as telomerase inhibitors that have relatively high potency or activity and that are orally bioavailable, and for compositions and methods for treating cancer and other diseases in which telomerase activity is present abnormally. The present invention meets these and other needs.
The present invention provides methods, compounds and compositions that are specific and effective for treating telomerase-mediated disorders, such as malignant conditions by targeting cells having telomerase activity. The methods, compounds, and compositions of the invention can be applied to a wide variety of malignant cell types and avoid the problems inherent in current cancer treatment modalities which are non-specific and excessively toxic.
In a first aspect, the present invention is based on the finding that certain known thiazolidinone compounds, as well as new thiazolidinone derivatives disclosed herein, are effective in the inhibition of telomerase enzyme activity, in vitro, ex vivo and in vivo. Thus, in certain aspects, the present invention provides methods of inhibiting telomerase by contacting telomerase with the compounds described herein. In particular embodiments, the telomerase to be inhibited is a mammalian telomerase, such as a human telomerase. A related aspect of the present invention is the discovery that thiazolidinone compounds inhibit the proliferation of cells that have telomerase activity, such as many cancer cells. Thus, this aspect of the present invention provides methods of inhibiting telomerase activity in a patient, preferably a mammal, suffering from a telomerase-mediated condition or disease, comprising administering to the patient a therapeutically effective amount of a telomerase inhibiting thiazolidinone compound, or a pharmaceutically acceptable salt thereof.
In another aspect, the present invention provides methods, compounds and compositions for inhibiting a telomerase enzyme, comprising contacting the telomerase enzyme with a composition or a compound of formula (I): 
Or its pharmaceutically acceptable salts, wherein X is oxygen or sulfur,  is a single or double bond; A is aryl or heteroaryl; R1 is hydrogen or lower alkyl; R2, R3 and R4 are independently selected from the group consisting of hydrogen, halo, alkyl, aryl, hydroxyl, alkoxyl, aryloxyl, aralkoxyl, cyano, nitro, alkylcarbamido, arylcarbamido, dialkylcarbamido, diarylcarbamido, alkylarylcarbamido, alkylthiocarbamido, arylthiocarbamido, dialkylthiocarbamido, diarylthiocarbamido, alkylarylthiocarbamido, amino, alkylamino, arylamino, dialkylamino, diarylamino, arylalkylamino, aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, dialkylaminocarbonyl, diarylaminocarbonyl, arylalkylaminocarbonyl, alkylcarbonyloxy, arylcarbonyloxy, carboxyl, alkoxycarbonyl, aryloxycarbonyl, sulfo, alkylsulfonylamido, arylsulfonamido, alkylsulfonyl, arylsulfonyl, alkylsulfinyl, arylsulfinyl and heteroaryl; L is a direct bond or a linking group having from 1 to 3 atoms independently selected from unsubstituted or substituted carbon, N, O or S; and n is 1 or 2.
In another aspect, the present invention provides methods, compounds and compositions for inhibiting a telomerase enzyme, comprising contacting the telomerase enzyme with a compound, or its pharmaceutically acceptable salt, having the formula (IV): 
where X is O or S;  is a single or double bond; R5 is H or lower alkyl; and Ar is a substituted or unsubstituted aryl, heteroaryl, aralkyl, heteroarylalkyl, arylalkeryl, heteroarylalkeryl, arylalkynyl or heteroarylalkynyl.
In another aspect, the present invention provides methods, compounds and compositions for inhibiting a telomerase enzyme, comprising contacting the telomerase enzyme with a composition or a compound of formula (V): 
or its pharmaceutically acceptable salt, wherein X is O or S; R6 is H or lower alkyl; W is CHxe2x95x90CH, S, or xe2x80x94Nxe2x95x90Cxe2x80x94; R7 is OH, halogen, mercapto, nitro, cyano, lower alkylthio, lower alkyl, lower alkoxy, lower alkanoyloxy, NR11R12 (wherein R11 and R12 are independently selected from the group consisting of hydrogen, lower alkyl, lower alkanoyl, aryl, heteroaryl, heteroarylalkyl, or R11 and R12 form a substituted or unsubstituted heterocyclic ring), CO2R13 (wherein R13 is selected from the group consisting of hydrogen, lower alkyl, aralkyl, and heteroarylalkyl), CONR11R12, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, aryloxy, heteroaryloxy, aralkyloxy, heteroarylalkyloxy, lower alkanoyl, aroyl, lower alkenyl, arylthio, or lower alkynyl; and when W represents S, R7 may also be H; L is O, S, SO, SO2, OCH2, SCH2, SOCH2, SO2CH2, or N(R10)(CH2)m (wherein R10 is substituted or unsubstituted aryl, heteroaryl, aralkyl, or heteroarylalkyl, and m is 0 or 1), (CH2)N(R10)(CH2)m, or CR13R14 (wherein R13 and R14 are independently selected from the group consisting of hydrogen, hydroxy, aryl, and heteroaryl); and A1 is cycloalkyl of formula (A1): 
wherein Z1 to Z5 are independently selected from the group consisting of hydrogen, lower alkyl, lower alkenyl, lower alkanoyloxy, mercapto, alkylthio, NR11R12; nitro, cyano, CO2R13, CONR11R12, aryl, heteroaryl, aryloxy, heteroaryloxy, aralkyloxy, heteroarylalkyloxy, halogen, and lower alkanoyl provided that when W is CHxe2x95x90CH, A may also by pyridyl.
The new compounds of the invention have many valuable uses as inhibitors of deleterious telomerase activity, such as, for example, in the treatment of cancer in mammals, such as humans. The pharmaceutical compositions of this invention can be employed in treatment regimens in which cancer cells are killed, in vivo, or can be used to kill cancer cells ex vivo. Thus, this invention provides therapeutic compounds and compositions for treating cancer, and methods for treating cancer and other telomerase-mediated conditions or diseases in humans and other mammals (e.g., cows, horses, sheep, steer, pigs and animals of veterinary interest such as cats and dogs).
Unless otherwise defined below, the terms used herein have their normally accepted scientific meanings. Definition of standard chemistry terms may be found in reference works, including Carey and Sundberg (1992) xe2x80x9cAdvanced Organic Chemistry 3rd Ed.xe2x80x9d Vols. A and B, Plenum Press, New York.
The term xe2x80x9cthiazolidinonexe2x80x9d or xe2x80x9cthiazolidinone derivativexe2x80x9d as used herein refers to compounds of the general formula: 
wherein X is O or S. When X is O, the derivatives are thiazolidinedione derivatives. When X is S, the derivatives are the thiazolidinonethione derivatives also known as rhodanines (see Examples 25-28, below).
The term xe2x80x9calkylxe2x80x9d as used herein refers to a straight, branched, or cyclic hydrocarbon chain fragment or radical containing between about one and about twenty carbon atoms, more preferably between about one and about ten carbon atoms (e.g., methyl, ethyl, n-propyl, iso-propyl, cyclopropyl, n-butyl, iso-butyl, tert-butyl, cyclobutyl, adamantyl, noradamantyl and the like). Straight, branched, or cyclic hydrocarbon chains having eight or fewer carbon atoms will also be referred to herein as xe2x80x9clower alkylxe2x80x9d. The hydrocarbon chains may further include one or more degrees of unsaturation, i.e., one or more double or triple bonds (e.g., vinyl, propargyl, allyl, 2-buten-1-yl, 2-cyclopenten-1-yl, 1,3-cyclohexadien-1-yl, 3-cyclohexen-1-yl and the like Alkyl groups containing double bonds such as just described will also be referred to herein as xe2x80x9calkenesxe2x80x9d. Similarly, alkyl groups having triple bonds will also be referred to herein as xe2x80x9calkynesxe2x80x9d. However, as used in context with respect to cyclic alkyl groups, the combinations of double and/or triple bonds do not include those bonding arrangements that render the cyclic hydrocarbon chain aromatic.
In addition, the term xe2x80x9calkylxe2x80x9d as used herein further includes one or more substitutions at one or more carbon atoms of the hydrocarbon fragment or radical. Such substitutions include, but are not limited to: aryl; heterocycle; halogen (to form, e.g., trifluoromethyl, xe2x80x94CF3); nitro (xe2x80x94NO2); cyano (xe2x80x94CN); hydroxyl (also referred to herein as xe2x80x9chydroxyxe2x80x9d), alkoxyl (also referred herein as alkoxy) or aryloxyl (also referred to herein as xe2x80x9caryloxyxe2x80x9d)(xe2x80x94OR); thio or mercapto, alkyl- or arylthio (xe2x80x94SR); amino, alkylamino, arylamino, dialkyl- or diarylamino, or arylalkylamino (xe2x80x94NRRxe2x80x2); aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, dialkylaminocarbonyl, diarylaminocarbonyl or arylalkylaminocarbonyl (xe2x80x94C(O)NRRxe2x80x2); carboxyl, or alkyl- or aryloxycarbonyl (xe2x80x94C(O)OR); carboxaldehyde, or aryl- or alkylcarbonyl (xe2x80x94C(O)R); iminyl, aryl- or alkyliminyl (xe2x80x94C(xe2x95x90NR)Rxe2x80x2); sulfo (xe2x80x94SO2OR); alkyl- or arylsulfonyl (xe2x80x94SO2R); ureido (xe2x80x94HNC(xe2x95x90O)NRRxe2x80x2); or thioureido (xe2x80x94HNC(xe2x95x90S)NRRxe2x80x2); where R and Rxe2x80x2 independently are hydrogen, aryl or alkyl as defined herein. Substituents including heterocyclic groups (i.e., heterocycle, heteroaryl, and heteroaralkyl) are defined by analogy to the above-described terms. For example, the term xe2x80x9cheterocycleoxyxe2x80x9d refers to the group xe2x80x94OR, where R is heterocycle as defined below.
The alkyl moiety of xe2x80x9clower alkanoylxe2x80x9d, xe2x80x9clower alkoxyxe2x80x9d, xe2x80x9clower alkanoyloxyxe2x80x9d, xe2x80x9clower alkylthioxe2x80x9d, is the same as xe2x80x9calkylxe2x80x9d defined above.
The term xe2x80x9cmethylenexe2x80x9d refers to the group xe2x80x94CH2xe2x80x94.
The term xe2x80x9cmethinexe2x80x9d refers to a methylene group for which one hydrogen atom has been replaced by a substituent as described above. The term xe2x80x9cmethinexe2x80x9d can also refer to a methylene group for which one hydrogen atom is replaced by bond to form an sp2-hybridized carbon center (i.e.,  greater than Cxe2x95x90O).
The term xe2x80x9chaloxe2x80x9d or xe2x80x9chalogenxe2x80x9d as used herein refers to the substituents fluoro, bromo, chloro, and iodo.
The term xe2x80x9ccarbonylxe2x80x9d as used herein refers to the functional group xe2x80x94C(O)xe2x80x94. However, it will be appreciated that this group may be replaced with well-known groups that have similar electronic and/or steric character, such as thiocarbonyl (xe2x80x94C(S)xe2x80x94); sulfinyl (xe2x80x94S(O)xe2x80x94); sulfonyl (xe2x80x94SO2xe2x80x94), phosphonyl (xe2x80x94PO2xe2x80x94), and methylidene (xe2x80x94C(xe2x95x90CH2)xe2x80x94). Other carbonyl equivalents will be familiar to those having skill in the medicinal and organic chemical arts.
The term xe2x80x9carylxe2x80x9d as used herein refers to cyclic aromatic, hydrocarbon chains having twenty or fewer carbon atoms, e.g., phenyl, naphthyl, biphenyl and anthracenyl. One or more carbon atoms of the aryl group may also be substituted with, e.g.: alkyl; aryl; heterocycle; formyl; halogen; nitro; cyano; hydroxyl, alkoxyl or aryloxyl; thio or mercapto, alkyl-, or arylthio; amino, alkylamino, arylamino, dialkyl-, diaryl-, or arylalkylamino; aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, dialkylaminocarbonyl, diarylaminocarbonyl or arylalkylaminocarbonyl; carboxyl, or alkyl- or aryloxycarbonyl; carboxaldehyde, or aryl- or alkylcarbonyl; iminyl, or aryl- or alkyliminyl; sulfo; alkyl- or arylsulfonyl; hydroximinyl, or aryl- or alkoximinyl; ureido; or thioureido. In addition, two or more alkyl or heteroalkyl substituents of an aryl group may be combined to form fused aryl-alkyl or aryl-heteroalkyl ring systems (e.g., tetrahydronaphthyl). Substituents including heterocyclic groups (e.g., heterocycleoxy, heteroaryloxy, and heteroaralkylthio) are defined by analogy to the above-described terms.
The term xe2x80x9caralkylxe2x80x9d as used herein refers to an aryl group that is joined to a parent structure by an alkyl group as described above, e.g., benzyl, xcex1-methylbenzyl, phenethyl, and the like. The aralkyl moiety of xe2x80x9caralkylsulfonylxe2x80x9d aralkyloxy is the same as xe2x80x9caralkylxe2x80x9d defined above.
The aryl moiety of xe2x80x9caroylxe2x80x9d, xe2x80x9carylalkenylxe2x80x9d, xe2x80x9carylalkenylxe2x80x9d, xe2x80x9carylsulfonylxe2x80x9d, xe2x80x9carylthioxe2x80x9d, xe2x80x9caryloxyxe2x80x9d, xe2x80x9carylalkenylsulfonylxe2x80x9d, xe2x80x9carylalkynylsulfonylxe2x80x9d is the same as xe2x80x9carylxe2x80x9d defined above.
The term xe2x80x9cheterocyclexe2x80x9d as used herein refers to a cyclic alkyl group or aryl group as defined above in which one or more carbon atoms have been replaced by a non-carbon atom, especially nitrogen, oxygen, or sulfur. Non-aromatic heterocycles will also be referred to herein as xe2x80x9ccyclic heteroalkylxe2x80x9d. Aromatic heterocycles are also referred to herein as xe2x80x9cheteroarylxe2x80x9d. For example, such groups include furyl, tetrahydrofuryl, pyrrolyl, pyrrolidinyl, thienyl, tetrahydrothienyl, oxazolyl, isoxazolyl, triazolyl, thiazolyl, isothiazolyl, pyrazolyl, pyrazolidinyl, oxadiazolyl, thiadiazolyl, imidazolyl, imidazolinyl, pyridyl, pyridazinyl, triazinyl, piperidinyl, morpholinyl, thiomorpholinyl, pyrazinyl, piperazinyl, pyrimidinyl, naphthyridinyl, benzofuranyl, benzothienyl, indolyl, indolinyl, indolizinyl, indazolyl, quinolizinyl, quinolinyl, isoquinolinyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, pteridinyl, quinuclidinyl, carbazolyl, acridiniyl, phenazinyl, phenothiazinyl, phenoxazinyl, purinyl, benzimidazolyl, benzthiazolyl, and benzoxazolyl.
The heteroaryl moiety of xe2x80x9cheteroarylalkylxe2x80x9d, xe2x80x9cheteroarylalkenylxe2x80x9d, xe2x80x9cheteroarylalkynylxe2x80x9d, xe2x80x9cheteroarylsulfonylxe2x80x9d, xe2x80x9cheteroarylalkylsulfonylxe2x80x9d, xe2x80x9cheteroarylalkenylsulfonylxe2x80x9d, xe2x80x9cheteroarylalkynylsulfonylxe2x80x9d, xe2x80x9cheteroaryloxyxe2x80x9d, xe2x80x9cheteroarylalkyloxyxe2x80x9d is the same as xe2x80x9cheteroarylxe2x80x9d defined above.
The above heterocyclic groups may further include one or more substituents at one or more carbon and/or non-carbon atoms of the heteroaryl group, e.g.: alkyl; aryl; heterocycle; halogen; nitro; cyano; hydroxyl, alkoxyl or aryloxyl; thio or mercapto, alkyl- or arylthio; amino, alkyl-, aryl-, dialkyl- diaryl-, or arylalkylamino; aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, dialkylaminocarbonyl, diarylaminocarbonyl or arylalkylaminocarbonyl; carboxyl, or alkyl- or aryloxycarbonyl; carboxaldehyde, or aryl- or alkylcarbonyl; iminyl, or aryl- or alkyliminyl; sulfo; alkyl- or arylsulfonyl; hydroximinyl, or aryl- or alkoximinyl; ureido; or thioureido. In addition, two or more alkyl substituents may be combined to form fused heterocycle-alkyl or heterocycle-aryl ring systems. Substituents including heterocyclic groups (e.g., heterocycleoxy, heteroaryloxy, and heteroaralkylthio) are defined by analogy to the above-described terms.
The term xe2x80x9cheterocyclealkylxe2x80x9d refers to a heterocycle group that is joined to a parent structure by one or more alkyl groups as described above, e.g., 2-piperidylmethyl, and the like. The term xe2x80x9cheteroaralkylxe2x80x9d as used herein refers to a heteroaryl group that is joined to a parent structure by one or more alkyl groups as described above, e.g., 2-thienylmethyl, and the like.
The compounds of the present invention may be used to inhibit or reduce telomerase enzyme activity and/or proliferation of cells having telomerase activity. In these contexts, inhibition and reduction of the enzyme or cell proliferation refers to a lower level of the measured activity relative to a control experiment in which the enzyme or cells are not treated with the test compound. In particular embodiments, the inhibition or reduction in the measured activity is at least a 10% reduction or inhibition. One of skill in the art will appreciate that reduction or inhibition of the measured activity of at least 20%, 50%, 75%, 90% or 100% may be preferred for particular applications.
As noted above, the immortalization of cells involves inter alia the activation of telomerase. More specifically, the connection between telomerase activity and the ability of many tumor cell lines, including skin, connective tissue, adipose, breast, lung, stomach, pancreas, ovary, cervix, uterus, kidney, bladder, colon, prostate, central nervous system (CNS), retina and blood tumor cell lines, to remain immortal has been demonstrated by analysis of telomerase activity (Kim et al.). This analysis, supplemented by data that indicates that the shortening of telomere length can provide the signal for replicative senescence in normal cells (see WO 93/23572), demonstrates that inhibition of telomerase activity can be an effective anti-cancer therapy. By xe2x80x9cinhibitionxe2x80x9d is simply meant a reagent, drug or chemical which is able to decrease the activity of the telomerase enzyme in vitro or in vivo. Such inhibitors can be readily identified using standard screening protocols in which a cellular extract or other preparation having telomerase activity is placed in contact with a potential inhibitor, and the level of telomerase activity measured in the presence or absence of the inhibitor, or in the presence of varying amounts of inhibitor. In this way, not only can useful inhibitors be identified, but the optimum level of such an inhibitor can be determined in vitro for further testing in vivo.
In a related aspect, the invention proves a method for inhibiting the ability of a cell to proliferate or replicate. In this method, one or more of the thiazolidinone compounds of the invention, that are capable of inhibiting telomerase enzyme activity, are provided during cell replication. As explained above, telomeres play a critical role in allowing the end of the linear chromosomal DNA to be replicated completely without the loss of terminal bases at the 5xe2x80x2-end of each strand. Immortal cells and rapidly proliferating cells use telomerase to add telomeric DNA repeats to chromosomal ends. Inhibition of telomerase will result in the proliferating cells not being able to add telomeres and they will eventually stop dividing. As will be evident to those of ordinary skill in the art, this method for inhibiting the ability of a cell to proliferate is useful for the treatment of a condition associated with an increased rate of proliferation of a cell, such as in cancer (telomerase-activity in malignant cells), and hematopoiesis (telomerase activity in hematopoietic stem cells), for example.
Thus, in one aspect, the present invention provides compositions and compounds for the prevention or treatment of many types of malignancies. In particular, the compounds of the present invention can provide a highly general method of treating many, if not most, malignancies, as demonstrated by the highly varied human tumor cell lines and tumors having telomerase activity. More importantly, the thiazolidinone compounds of the present invention can be effective in providing treatments that discriminate between malignant and normal cells to a high degree, avoiding many of the deleterious side-effects present with most current chemotherapeutic regimes which rely on agents that kill dividing cells indiscriminately. Representative known thiazolidinedione compounds include the glitazones, such as, for example, troglitazone (also known as CS-045 (Sankyo) and CI-991 (Park-Davis)), pioglitazone (also known as AD-4833 and U-72107E), rosiglitazone (also known as BRL49653), englitazone (also known as CP-68,722), and ciglitazone.
In another aspect, the present invention provides new compounds, pharmaceutical compositions and methods relating to the new compounds, or their pharmaceutically acceptable salts, for inhibiting a telomerase enzyme, comprising contacting the telomerase enzyme with a compound, or its pharmaceutically acceptable salt, having the formula (I): 
wherein X is oxygen or sulfur,  is a single or double bond; A is aryl or heteroaryl; R1 is hydrogen or lower alkyl; R2, R3 and R4 are independently selected from the group consisting of hydrogen, halo, alkyl, aryl, hydroxyl, alkoxyl, aryloxyl, aralkoxyl, cyano, nitro, alkylcarbamido, arylcarbamido, dialkylcarbamido, diarylcarbamido, alkylarylcarbamido, alkylthiocarbamido, arylthiocarbamido, dialkylthiocarbamido, diarylthiocarbamido, alkylarylthiocarbamido, amino, alkylamino, arylamino, dialkylamino, diarylamino, arylalkylamino, aminocarbonyl, alkylamino-carbonyl, arylaminocarbonyl, dialkylaminocarbonyl, diarylaminocarbonyl, arylalkylamino-carbonyl, alkylcarbonyloxy, arylcarbonyloxy, carboxyl, alkoxycarbonyl, aryloxycarbonyl, sulfo, alkylsulfonylamido, arylsulfonamido, alkylsulfonyl, arylsulfonyl, alkylsulfinyl, arylsulfinyl and heteroaryl; L is a direct bond or a linking group having from 1 to 3 atoms independently selected from unsubstituted or substituted carbon, N, O or S; and n is 1 or 2.
In certain embodiments, the new compounds of the present invention have the general structure shown as formula II below: 
and their pharmaceutically acceptable salts, wherein X is O or S, and R2, R3, R4, L, and n are as defined above.
In the compounds of formula I, A may be aryl to form, for example, a phenyl moiety. Alternatively, A may be heteroaryl, such as, for example, pyridine, quinoline, isoquinoline, thiophene, furan, imidazole, benzimidazole, pyrazole, and the like. In presently preferred embodiments, A is phenyl, as in formula (II). In other presently preferred embodiments, when n is 1, R1 can not be hydrogen. In yet other presently preferred embodiments, at least one of R2 and R3 is other than hydrogen. In presently particularly preferred embodiments, at least one of R2 and R3 is halo, and most preferably both R2 and R3 are halo to form a dihalo-substituted phenyl moiety.
As noted above, L may be a direct bond, or may be a 1 to 3 atom linking group wherein the atoms of the linking group independently selected from unsubstituted or substituted carbon, N, O or S. Representative linking groups useful in the compounds of the invention include, for example xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94CH2, xe2x80x94OCH2xe2x80x94, xe2x80x94OC(O)xe2x80x94, xe2x80x94C02xe2x80x94, xe2x80x94NHC(O)xe2x80x94, xe2x80x94C(O)NHxe2x80x94, xe2x80x94OC(O)CH2xe2x80x94, xe2x80x94OC(O)NHxe2x80x94, and xe2x80x94NHC(O)NHxe2x80x94.
For purposes of illustration, representative compounds include, without limitation, 5-(2-(3,4-Dichlorophenyl)benzylidene)thiazolidine-2,4-dione, 5-(3-(3,4-Dichlorophenyl)benzylidene)thiazolidine-2,4-dione, 5-(4-(3,4-Dichlorobenzyloxy)benzylidene)thiazolidine-2,4-dione, 5-(2-(3,4-Dichlorobenzyloxy)benzylidene)thiazolidine-2,4-dione, 5-(4-(3,4-Dichlorobenzamido)benzylidene)thiazolidine-2,4-dione, 5-(4-(N-3,4-Dichlorophenyureido)-benzylidene)thiazolidine-2,4-dione, 5-(2-(N-3,4-Dichlorophenyureido)benzylidene)-thiazolidine-2,4-dione, 5-(2-(N-3,4-Dichlorophenylcarbamido)benzylidene)thiazolidine-2,4-dione, 5-(3-(N-3,4-Dichlorophenylcarbarmido)benzylidene)thiazolidine-2,4-dione, 5-(4-(N-3,4-Dichlorophenylcarbamido)benzylidene)thiazolidine-2,4-dione, 5-(4-(N-3,4-Dichlorophenylcarbamoyloxy)benzylidene)thiazolidine-2,4-dione, 5-(4-(3,4-Dichlorophenoxycarbonyl)benzylidene)thiazolidine-2,4-dione, 5-(2-(3,4-Dichlorophenoxycarbonyl)benzylidene)thiazolidine-2,4-dione, 5-(2-(3,4-Dichlorophenylacetoxy)benzylidene)thiazolidine-2,4-dione, 5-(3-(3,4-Dichlorophenylacetoxy)-benzylidene)thiazolidine-2,4-dione, 5-(4-(3,4-Dichlorophenylacetoxy)benzylidene)thiazolidine-2,4-dione, 5-(2-(3,4-Dichlorobenzoyloxy)benzylidene)thiazolidine-2,4-dione, 5-(3-(3,4-Dichlorobenezoyloxy)-benzylidene)thiazolidine-2,4-dione, 5-(4-(3,4-Dichlorobenzoyloxy)-benzylidene)thiazolidine-2,4-dione, 5-(3,4-Bis-(3,4-dichlorobenzyloxy)benzylidine)thiazolidine-2,4-dione, 5-(2-(3,4-Dichlorophenoxy)benzylidine)thiazolidine-2,4-dione, 5-(4-(3,4-Dichlorophenoxy)benzylidine)thiazolidine-2,4-dione, 5-(2,5-Bis-(3,4-dichlorobenzyloxy)-benzylidine)thiazolidine-2,4-dione, 5-(2,4-Bis-(3,4-dichlorobenzyloxy)benzylidine)-thiazolidine-2,4-dione, 5-(2-(3,4-Dichlorobenylthio)-3H-pyrimidin-4-on-6-ylmethylidene)-rhodanine, 5-(2-(3,4-Dichlorobenzylthio)pyrimidin-4-ylmethylidene)rhodanine, 5-(2-(3,4-Dichlorobenzylthio)pyrimidin-4-ylmethylidene)rhodanine, 5-(3-Cyano-2-(3,4-dichlorobenzylthio)pyridin-6-ylmethylidene)thiazolidine-2,4-dione and 5-(3-(3,4-Dichlorobenzyloxy)benzylidene)thiazolidine-2,4-dione.
In certain embodiments, the new compounds of the present invention have the general structure shown as formula III below: 
and their pharmaceutically acceptable salts, wherein X is O or S, and R1, R2, R3, R4, and L are as defined above.
In another embodiment, the present invention provides methods, compounds and compositions for inhibiting a telomerase enzyme, comprising contacting the telomerase enzyme with a compound, or its pharmaceutically acceptable salt, having the formula (IV): 
where X is O or S;  is a single or double bond; R5 is H or lower alkyl; and Ar is a substituted or unsubstituted aryl, heteroaryl, aralkyl, heteroarylalky, arylalkenyl, heteroarylalkenyl, arylalkynyl or heteroarylalkynyl.
Compounds of formula (IV) having the double bond can be obtained by reacting a thiazolidine derivative with an aromatic carbonyl compound. The reaction can be carried out optionally in the presence of a base catalyst and optionally in a solvent. The base catalyst, usually present in about 0.1 to about 1 equivalent, may be piperidine, piperidinium acetate, diethylamine, pyridine, sodium acetate, potassium carbonate, sodium carbonate, and the like. The solvent may be an alcohol, such as methanol, ethanol, propanol, or the like, an ether, such as diethyl ether, tetrahydrofuran, dioxane, or the like, or a hydrocarbon, such as benzene, roluene, xylene, or the like, and mixtures thereof. The reaction is carried out at a temperature of about room temperature to about 200xc2x0 C., preferably about 50-100xc2x0 C., and completes in about one hour to about 50 hours. Compounds of formula (IV) wherein  is a single bond can be synthesized by reducing the double bond of the compound made above. Typically, hydrogenation is carried out using a noble metal catalyst, such as palladium, platinum, rhodium, or the like, as is well known in the art.
In another aspect, the present invention provides methods, compounds and compositions for inhibiting a telomerase enzyme, comprising contacting the telomerase enzyme with a compound, or its pharmaceutically acceptable salt, having the formula (V): 
wherein X is O or S; R6 is H or lower alkyl; W is CHxe2x95x90CH, S, or xe2x80x94Nxe2x95x90Cxe2x80x94; R7 is H, OH, halogen, mercapto, nitro, cyano, lower alkylthio, lower alkyl, lower alkoxy, lower alkanoyloxy, NR11R12 (wherein R11 and R12 are independently selected from the group consisting of hydrogen, lower alkyl, lower alkanoyl, aryl, heteroaryl, heteroarylalkyl, or R11 and R12 form a substituted or unsubstituted heterocyclic ring), CO2R13 (wherein R13 is selected from the group consisting of hydrogen, lower alkyl, aralkyl, and heteroarylalkyl), CONR11R12, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, aryloxy, heteroaryloxy, aralkyloxy, heteroarylalkyloxy, lower alkanoyl, aroyl, lower alkenyl, arylthio, or lower alkynyl; L is O, S, SO, SO2, OCH2, SCH2, SOCH2, SO2CH2, or N(R10)(CH2)m (wherein R10 is substituted or unsubstituted aryl, heteroaryl, aralkyl, or heteroarylalkyl, and m is 0 or 1), (CH2)N(R10)(CH2)m, or CR13R14 (wherein R13 and R14 are independently selected from the group consisting of hydrogen, hydroxy, aryl, and heteroaryl); and A1 is cycloarlkyl of formula (A1): 
wherein Z1 to Z5 are independently selected from the group consisting of hydrogen, lower alkyl, lower alkenyl, lower alkanoyloxy, mercapto, alkylthio, NR11R12, nitro, cyano, CO2R13, CONR11R12, aryl, heteroaryl, aryloxy, heteroaryloxy, aralkyloxy, heteroarylalkyloxy, halogen, and lower alkanoyl provided that when W is CHxe2x95x90CH, A1 may also by pyridyl.
Examples of the compound of the present invention represented by formulae I to V are shown in Tables 1 to 6 below, though compounds of the present invention are not restricted thereby.
The compounds of the present invention can be synthesized using techniques and materials known to those of skill in the art, such as described, for example, in March, ADVANCED ORGANIC CHEMISTRY 4th Ed., (Wiley 1992); Carey and Sundberg, ADVANCED ORGANIC CHEMISTRY 3rd Ed., Vols. A and B (Plenum 1992), and Green and Wuts, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS 2nd Ed. (Wiley 1991). Starting materials for the compounds of the invention may be obtained using standard techniques and commercially available precursor materials, such as those available from Aldrich Chemical Co. (Milwaukee, Wis.), Sigma Chemical Co. (St. Louis, Miss.), Lancaster Synthesis (Windham, N.H.), Apin Chemicals, Ltd. (New Brunswick, N.J.), Ryan Scientific (Columbia, S.C.), Maybridge (Cornwall, England), Arcos (Pittsburgh, Pa.), and Trans World Chemicals (Rockville, Md.).
The procedures described herein for synthesizing the compounds of the invention may include one or more steps of protection and deprotection (e.g., the formation and removal of acetal groups). In addition, the synthetic procedures disclosed below can include various purifications, such as column chromatography, flash chromatography, thin-layer chromatography (TLC), recrystallization, distillation, high-pressure liquid chromatography (HPLC) and the like. Also, various techniques well known in the chemical arts for the identification and quantification of chemical reaction products, such as proton and carbon-13 nuclear magnetic resonance (1H and 13C NMR), infrared and ultraviolet spectroscopy (IR and UV), X-ray crystallography, elemental analysis (EA), HPLC and mass spectroscopy (MS) can be used as well. Methods of protection and deprotection, purification, identification and quantification are well known in the chemical arts.
Compounds of the class represented by formulas I, II and III can be synthesized using General Procedure 1 and General Procedure 2 described in detail in the Examples below. Detailed protocols from which the individual compounds described above can be synthesized are also provided in the Examples. The compounds of formula IV, where L is SO or SO2 can be synthesised by oxidizing the corresponsing S compound in an inert solvent. The inert solvent may be dichloromethane, methanol, tetrahydrofuran, ether, hexane, toluene, cyclohexane, or the like, and mixtures thereof. The oxidizing agent may be m-chloroperbenzoic acid, hydrogen peroxide, or the like. The reaction is carried out at a temperature in the range of about xe2x88x9278xc2x0 C. to the boiling point of the solvent, preferably from about 0xc2x0 C. to about 30xc2x0 C. for about 0.5 to about 12 hours.
The compounds of the present invention demonstrate inhibitory activity against telomerase activity in vivo, as has been and can be demonstrated as described below. The in vitro activities of the compounds of the invention can also be demonstrated using the methods described herein. As used herein, the term xe2x80x9cex vivoxe2x80x9d refers to tests performed using living cells in tissue culture.
One method used to identify compounds of the invention that inhibit telomerase activity involves placing cells, tissues, or preferably a cellular extract or other preparation containing telomerase in contact with several known concentrations of a test compound in a buffer compatible with telomerase activity. The level of telomerase activity for each concentration of test compound is measured and the IC50 (the concentration of the test compound at which the observed activity for a sample preparation was observed to fall one-half of its original or a control value) for the compound is determined using standard techniques. Other methods for determining the inhibitory concentration of a compound of the invention against telomerase can be employed as will be apparent to those of skill in the art based on the disclosure herein.
With the above-described methods, IC50 values for several of the compounds of the present invention were determined, and found to be below 100 xcexcM.
With respect to the treatment of malignant diseases using the compounds described herein, compounds of the present invention are expected to induce crisis in telomerase-positive cell lines. Treatment of telomerase-positive cell lines, such as HEK-293 and HeLa cells, with a compound of the invention is also expected to induce a reduction of telomere length in the treated cells.
Compounds of the invention are also expected to induce telomere reduction during cell division in human tumor cell lines, such as the ovarian tumor cell lines OVCAR-5 and SK-OV-3. Importantly, however, in normal human cells used as a control, such as BJ cells of fibroblast origin, the observed reduction in telomere length is expected to be no different from cells treated with a control substance, e.g., dimethyl sulfoxide (DMSO). The compounds of the invention also are expected to demonstrate no significant cytotoxic effects at concentrations below about 5 xcexcM in the normal cells.
In addition, the specificity of the compounds of the present invention for telomerase can be determined by comparing their activity (IC50) with respect to telomerase to other enzymes having similar nucleic acid binding or modifying activity similar to telomerase in vitro. Such enzymes include DNA Polymerase I, HeLa RNA Polymerase II, T3 RNA Polymerase, MMLV Reverse Transcriptase, Topoisomerase I, Topoisomerase II, Terminal Transferase and Single-Stranded DNA Binding Protein (SSB). Compounds having lower IC50 values for telomerase as compared to the IC50 values toward the other enzymes being screened are said to possess specificity for telomerase.
In vivo testing can also be performed using a mouse xenograft model, for example, in which OVCAR-5 tumor cells are grafted onto nude mice, in which mice treated with a compound of the invention are expected to have tumor masses that, on average, may increase for a period following the initial dosing, but will begin to shrink in mass with continuing treatment. In contrast, mice treated with a control (e.g., DMSO) are expected to have tumor masses that continue to increase.
From the foregoing those skilled in the art will appreciate that the present invention also provides methods for selecting treatment regimens involving administration of a compound of the invention. For such purposes, it may be helpful to perform a terminal restriction fragment (TRF) analysis in which DNA from tumor cells is analyzed by digestion with restriction enzymes specific for sequences other than the telomeric (T2 AG3)N sequence. Following digestion of the DNA, gel electrophoresis is performed to separate the restriction fragments according to size. The separated fragments are then probed with nucleic acid probes specific for telomeric sequences to determine the lengths of the terminal fragments containing the telomere DNA of the cells in the sample. By measuring the length of telomeric DNA, one can estimate how long a telomerase inhibitor should be administered and whether other methods of therapy (e.g., surgery, chemotherapy and/or radiation) should also be employed. In addition, during treatment, one can test cells to determine whether a decrease in telomere length over progressive cell divisions is occurring to demonstrate treatment efficacy.
The present invention also provides pharmaceutical compositions for inhibiting cell proliferation of telomerase positive cells, and treating cancer and other conditions in which inhibition of telomerase is an effective therapy. These compositions include a therapeutically effective amount of a telomerase inhibiting compound of the invention in a pharmaceutically acceptable carrier or salt.
In one embodiment, the present invention provides methods, compounds and compositions for inhibiting a telomerase enzyme, inhibiting proliferation of telomerase positive cells, and for treating cancer in a mammal. The compositions of the invention include a therapeutically effective amount of a compound of formulas I to V (or a pharmaceutically acceptable salt thereof) in a pharmaceutically acceptable carrier. The compounds and compositions of the present invention may also be used for the treatment of other telomerase mediated conditions or diseases, such as, for example, other hyperproliferative or autoimmune disorders such as psoriasis, rheumatoid arthritis, immune system disorders requiring immune system suppression, immune system reactions to poison ivy or poison oak, and the like.
In addition, it will be appreciated that therapeutic benefits for treatment of cancer can be realized by combining a telomerase inhibitor of the invention with other anti-cancer agents, including other inhibitors of telomerase such as described in U.S. Pat. Nos. 5,656,638, 5,760,062, 5,767,278, 5,770,613 and 5,863,936. The choice of such combinations will depend on various factors including, but not limited to, the type of disease, the age and general health of the patient, the aggressiveness of disease progression, the TRF length and telomerase activity of the diseased cells to be treated and the ability of the patient to tolerate the agents that comprise the combination. For example, in cases where tumor progression has reached an advanced state, it may be advisable to combine a telomerase inhibiting compound of the invention with other agents and therapeutic regimens that are effective at reducing tumor size (e.g. radiation, surgery, chemotherapy and/or hormonal treatments). In addition, in some cases it may be advisable to combine a telomerase inhibiting agent of the invention with one or more agents that treat the side effects of a disease, e.g., an analgesic, or agents effective to stimulate the patient""s own immune response (e.g., colony stimulating factor).
In one such method, a pharmaceutical formulation comprises a telomerase inhibitor of the invention with an anti-angiogenesis agent, such as fumagillin, fumagillin derivatives, or AGM-1470. The latter compound is available from Takeda Chemical Industries, Ltd., while the former compounds are described in Ingber, et al., Dec. 6, 1990, xe2x80x9cSynthetic analogues of fumagillin that inhibit angiogenesis and suppress tumor growthxe2x80x9d, Nature 348:555-557. Other combinations may include, but are not limited to, a telomerase inhibitor of the invention in addition to one or more antineoplastic agents or adjuncts (e.g., folinic acid or mesna).
Antineoplastic agents suitable for combination with the compounds of the present invention include, but are not limited to, alkylating agents including alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines, such as a benzodizepa, carboquone, meturedepa and uredepa; ethylenimines and methylmelamines such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolmelamine; nitrogen mustards such as chlorambucil, chlornaphazine, cyclophosphamide, estramustine, iphosphamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichine, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitroso ureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimustine. Additional agents include dacarbazine, mannomustine, mitobronitol, mitolactol and pipobroman. Still other classes of relevant agents include antibiotics, hormonal antineoplastics and antimetabolites. Yet other combinations will be apparent to those of skill in the art.
Additional agents suitable for combination with the compounds of the present invention include protein synthesis inhibitors such as abrin, aurintricarboxylic acid, chloramphenicol, colicin E3, cycloheximide, diphtheria toxin, edeine A, emetine, erythromycin, ethionine, fluoride, 5-fluorotryptophan, fusidic acid, guanylyl methylene diphosphonate and guanylyl imidodiphosphate, kanamycin, kasugamycin, kirromycin, and O-methyl threonine. Additional protein synthesis inhibitors include modeccin, neomycin, norvaline, pactamycin, paromomycine, puromycin, ricin, _-sarcin, shiga toxin, showdomycin, sparsomycin, spectinomycin, streptomycin, tetracycline, thiostrepton and trimethoprim. Inhibitors of DNA synthesis, including alkylating agents such as dimethyl sulfate, mitomycin C, nitrogen and sulfur mustards, MNNG and NMS; intercalating agents such as acridine dyes, actinomycins, adriamycin, anthracenes, benzopyrene, ethidium bromide, propidium diiodide-intertwining, and agents such as distamycin and netropsin, can also be combined with compounds of the present invention in pharmaceutical compositions. DNA base analogs such as acyclovir, adenine xcex2-1-D-arabinoside, amethopterin, aminopterin, 2-aminopurine, aphidicolin, 8-azaguanine, azaserine, 6-azauracil, 2xe2x80x2-azido-2xe2x80x2-deoxynucleosides, 5-bromodeoxycytidine, cytosine xcex2-1-D-arabinoside, diazooxynorleucine, dideoxynucleosides, 5-fluorodeoxycytidine, 5-fluorodeoxyuridine, 5-fluorouracil, hydroxyurea and 6-mercaptopurine also can be used in combination therapies with the compounds of the invention. Topoisomerase inhibitors, such as coumermycin, nalidixic acid, novobiocin and oxolinic acid, inhibitors of cell division, including colcemide, colchicine, vinblastine and vincristine; and RNA synthesis inhibitors including actinomycin D, xcex1-amanitine and other fungal amatoxins, cordycepin (3xe2x80x2-deoxyadenosine), dichlororibofuranosyl benzimidazole, rifampicine, streptovaricin and streptolydigin also can be combined with the compounds of the invention to provide pharmaceutical compositions.
In another embodiment, the present invention includes compounds and compositions in which a telomerase inhibitor is either combined with or covalently bound to a cytotoxic agent bound to a targeting agent, such as a monoclonal antibody (e.g., a murine or humanized monoclonal antibody). It will be appreciated that the latter combination may allow the introduction of cytotoxic agents into cancer cells with greater specificity. Thus, the active form of the cytotoxic agent (i.e., the free form) will be present only in cells targeted by the antibody. Of course, the telomerase inhibitors of the invention may also be combined with monoclonal antibodies that have therapeutic activity against cancer.
In addition to the application of the telomerase inhibitors of the present invention to the treatment of mammalian diseases characterized by telomerase activity, telomerase inhibitors such as those disclosed herein, can be applied to agricultural phytopathogenic organisms that are characterized by telomerase activity. These organisms include nematodes such as Ceanorhabditis elegans, in which telomerase activity has been found, and in fungi which are expected to have telomerase activity based on the determination that the DNA of the fungus Ustilago maydis exhibits telomeres having the tandem TTAGGG (SEQ ID NO:1) repeats that are maintained by telomerase. Also, protozoans have TTAGGG (SEQ ID NO:1) telomeres and cause human disease. The telomerase-inhibiting compounds of the invention can be administered to plants and soil infected with phytopathogenic organisms having telomerase activity alone, or in combination with other telomerase-inhibiting agents and/or other agents used to control plant diseases. The determination of the compositions used to control such phytopathogenic organisms and the appropriate modes of delivering such compositions will be known to those having skill in the agricultural arts.
The determination that nematodes, protozoans and possibly fungi have telomerase activity also indicates that the telomerase inhibitors provided by the present invention can be used to treat nematode infections in humans and animals of veterinary interest such as dogs and cats. Nematode infection in humans and animals often is in the form of hookworm or roundworm infection and leads to a host of deadly secondary illnesses such as meningitis, myocarditis, and various neurological diseases. Thus, it will be appreciated that administration of the telomerase-inhibiting compounds such as those of the invention, alone, or in combination with other telomerase-inhibiting agents and/or other therapeutic agents, can be used to control nematode, protozoan and fungal infections in humans and animals.
In general, a suitable effective dose of a compound of the invention will be in the range of 0.001 to 1000 milligram (mg) per kilogram (kg) of body weight of the recipient per day, preferably in the range of 0.001 to 100 mg per kg of body weight per day, more preferably between about 0.1 and 100 mg per kg of body weight per day and still more preferably in the range of between 0.1 to 10 mg per kg of body weight per day. The desired dosage is preferably presented in one, two, three, four, or more subdoses administered at appropriate intervals throughout the day, or by the action of a continuous pump. These subdoses can be administered as unit dosage form, for example, containing 5 to 10,000 mg, preferably 10 to 1000 mg of active ingredient per unit dosage from. Preferably, the dosage is presented once per day at a dosing at least equal to TID, or is administered using a continuous pump delivery system.
The composition used in these therapies can be in a variety of forms. These include, for example, solid, semi-solid, and liquid dosage forms, such as tablets, pills, powders, liquid solutions or suspensions, liposomes, and injectable and infusible solutions. The preferred form depends on the intended mode of administration and therapeutic application. The compositions also preferably include conventional pharmaceutically acceptable carriers and adjuvants, as is well known to those of skill in the art. See, e.g., REMINGTON""S PHARMACEUTICAL SCIENCES, Mack Publishing Co.: Easton, Pa., 17th Ed. (1985). Preferably, administration will be by oral or parenteral (including subcutaneous, intramuscular, intravenous, and intradernal) routes. More preferably, the route of administration will be oral. The therapeutic methods and agents of this invention can of course be used concomitantly or in combination with other methods and agents for treating a particular disease or disease condition.
While it is possible to administer the active ingredient of this invention alone, it is preferable to present a therapeutic agent as part of a pharmaceutical formulation or composition. The formulations of the present invention comprise at least one telomerase activity-inhibiting compound of this invention in a therapeutically or pharmaceutically effective dose together with one or more pharmaceutically or therapeutically acceptable carriers and optionally other therapeutic ingredients. Various considerations for preparing such formulations are described, e.g., in Gilman et al. (eds.) GOODMAN AND GILMAN""S: THE PHARMACOLOGICAL BASES OF THERAPEUTICS, 8th Ed., Pergamon Press (1990); and REMINGTON""S supra. Methods for administration are discussed therein, e.g., for oral, intravenous, intraperitoneal, intramuscular, and other forms of administration. Typically, methods for administering pharmaceutical compositions will be either topical, parenteral, or oral administration methods for prophylactic and/or therapeutic treatment. Oral administration is preferred. The pharmaceutical compositions can be administered in a variety of unit dosage forms depending upon the method of administration. As noted above, unit dosage forms suitable for oral administration include powders, tablets, pills, and capsules.
One can use topical administration to deliver a compound of the invention by percutaneous passage of the drug into the systemic circulation of the patient. The skin sites include anatomic regions for transdermally administering the drug, such as the forearm, abdomen, chest, back, buttock, and mastoidal area. The compound is administered to the skin by placing on the skin either a topical formulation comprising the compound or a transdermal drug delivery device that administers the compound. In either embodiment, the delivery vehicle is designed, shaped, sized, and adapted for easy placement and comfortable retention on the skin.
A variety of transdermal drug delivery devices can be employed with the compounds of this invention. For example, a simple adhesive patch comprising a backing material and an acrylate adhesive can be prepared. The drug and any penetration enhancer can be formulated into the adhesive casting solution. The adhesive casting solution can be cast directly onto the backing material or can be applied to the skin to form an adherent coating. See, e.g., U.S. Pat. Nos. 4,310,509; 4,560,555; and 4,542,012.
In other embodiments, the compound of the invention will be delivered using a liquid reservoir system drug delivery device. These systems typically comprise a backing material, a membrane, an acrylate based adhesive, and a release liner. The membrane is sealed to the backing to form a reservoir. The drug or compound and any vehicles, enhancers, stabilizers, gelling agents, and the like are then incorporated into the reservoir. See, e.g., U.S. Pat. Nos. 4,597,961; 4,485,097; 4,608,249; 4,505,891; 3,843,480; 3,948,254; 3,948,262; 3,053,255; and 3,993,073.
Matrix patches comprising a backing, a drug/penetration enhancer matrix, a membrane, and an adhesive can also be employed to deliver a compound of the invention transdermally. The matrix material typically will comprise a polyurethane foam. The drug, any enhancers, vehicles, stabilizers, and the like are combined with the foam precursors. The foam is allowed to cure to produce a tacky, elastomeric matrix which can be directly affixed to the backing material. See, e.g., U.S. Pat. Nos. 4,542,013; 4,460,562; 4,466,953; 4,482,534; and 4,533,540.
Also included within the invention are preparations for topical application to the skin comprising a compound of the invention, typically in concentrations in the range from about 0.001% to 10%, together with a non-toxic, pharmaceutically acceptable topical carrier. These topical preparations can be prepared by combining an active ingredient according to this invention with conventional pharmaceutical diluents and carriers commonly used in topical dry, liquid, and cream formulations. Ointment and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents. Such bases may include water and/or an oil, such as liquid paraffin or a vegetable oil, such as peanut oil or castor oil. Thickening agents that may be used according to the nature of the base include soft paraffin, aluminum stearate, cetostearyl alcohol, propylene glycol, polyethylene glycols, woolfat, hydrogenated lanolin, beeswax, and the like.
Lotions may be formulated with an aqueous or oily base and will, in general, also include one or more of the following: stabilizing agents, emulsifying agents, dispersing agents, suspending agents, thickening agents, coloring agents, perfumes, and the like. Powders may be formed with the aid of any suitable powder base, e.g., talc, lactose, starch, and the like. Drops may be formulated with an aqueous base or non-aqueous base also comprising one or more dispersing agents, suspending agents, solubilizing agents, and the like. Topical administration of compounds of the invention may also be preferred for treating diseases such as skin cancer and fungal infections of the skin (pathogenic fungi typically express telomerase activity).
The topical pharmaceutical compositions according to this invention may also include one or more preservatives or bacteriostatic agents, e.g., methyl hydroxybenzoate, propyl hydroxybenzoate, chlorocreosol, benzalkonium chlorides, and the like. The topical pharmaceutical compositions also can contain other active ingredients such as antimicrobial agents, particularly antibiotics, anesthetics, analgesics, and antipruritic agents.
The compounds of the present invention can also be delivered through mucosal membranes. Transmucosal (i.e., sublingual, buccal, and vaginal) drug delivery provides for an efficient entry of active substances to systemic circulation and reduces immediate metabolism by the liver and intestinal wall flora. Transmucosal drug dosage forms (e.g., tablet, suppository, ointment, pessary, membrane, and powder) are typically held in contact with the mucosal membrane and disintegrate and/or dissolve rapidly to allow immediate systemic absorption. Note that certain such routes may be used even where the patient is unable to ingest a treatment composition orally. Note also that where delivery of a telomerase inhibitor of the invention would be enhanced, one can select a composition for delivery to a mucosal membrane, e.g., in cases of colon cancer one can use a suppository to deliver the telomerase inhibitor.
For delivery to the buccal or sublingual membranes, typically an oral formulation, such as a lozenge, tablet, or capsule, will be used. The method of manufacture of these formulations is known in the art, including, but not limited to, the addition of the pharmacological agent to a pre-manufactured tablet; cold compression of an inert filler, a binder, and either a pharmacological agent or a substance containing the agent (as described in U.S. Pat. No. 4,806,356); and encapsulation. Another oral formulation is one that can be applied with an adhesive, such as the cellulose derivative hydroxypropyl cellulose, to the oral mucosa, for example as described in U.S. Pat. No. 4,940,587. This buccal adhesive formulation, when applied to the buccal mucosa, allows for controlled release of the pharmacological agent into the mouth and through the buccal mucosa.
Parenteral administration is generally characterized by injection, either subcutaneously, intramuscularly, or intravenously. Thus, this invention provides compositions for intravenous administration that comprise a solution of a compound of the invention dissolved or suspended in an acceptable carrier. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Suitable excipients are, for example, water, buffered water, saline, dextrose, glycerol, ethanol, or the like. These compositions will be sterilized by conventional, well known sterilization techniques, such as sterile filtration. The resulting solutions can be packaged for use as is or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration. In addition, if desired, the pharmaceutical compositions to be administered may also contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, pH buffering agents and the like, such as for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, etc. Such formulations will be useful in treating ovarian cancers.
Another method of parenteral administration employs the implantation of a slow-release or sustained-release system, such that a constant level of dosage is maintained. See, e.g., U.S. Pat. No. 3,710,795.
Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, etc., an active compound as defined above and optional pharmaceutical adjuvants in an excipient, such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, olive oil, and other lipophilic solvents, and the like, to form a solution or suspension. If desired, the pharmaceutical composition to be administered may also contain minor amounts of nontoxic auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, and the like, for example, sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, etc. Actual methods of preparing such dosage forms are known and will be apparent to those skilled in this art; for example, see REMINGTON""S PHARMACEUTICAL SCIENCES, supra. The composition or formulation to be administered will contain an effective amount of an active compound of the invention.
For solid compositions, conventional nontoxic solid carriers can be used and include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For oral administration, a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 0.1-95% of active ingredient, preferably about 20%.
The compositions containing the compounds of the invention can be administered for prophylactic and/or therapeutic treatments. In therapeutic applications, compositions are administered to a patient already suffering from a disease, as described above, in an amount sufficient to cure or at least partially arrest the symptoms of the disease and its complications. An amount adequate to accomplish this is defined as a xe2x80x9ctherapeutically effective amount or dose.xe2x80x9d Amounts effective for this use will depend on the severity of the disease and the weight and general state of the patient.
In addition to internal (in vivo) administration, the compounds and compositions of the invention may be applied ex vivo to achieve therapeutic effects, as for example, in the case of a patient suffering from leukemia. In such an application, cells to be treated, e.g., blood or bone marrow cells, are removed from a patient and treated with a pharmaceutically effective amount of a compound of the invention. The cells are returned to the patient following treatment. Such a procedure can allow for exposure of cells to concentrations of therapeutic agent for longer periods or at higher concentrations than otherwise available.
Once improvement of the patient""s conditions has occurred, as, for example, by the occurrence of remission in the case of a cancer patient, a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, can be reduced, as a function of the systems, to a level at which the improved condition is retained. When the symptoms have been alleviated to the desired level, treatment can cease. Patients can, however, require additional treatment upon any recurrence of the disease symptoms.
In prophylactic applications (e.g. chemoprevention), compositions containing the compounds of the invention are administered to a patient susceptible to or otherwise at risk of a particular disease. Such an amount is defined to be a xe2x80x9cprophylactically effective amount or dose.xe2x80x9d In this use, the precise amounts again depend on the patient""s state of health and weight.
As will be apparent to those of skill in the art upon reading of this disclosure, the present invention provides valuable reagents relating to human and mammalian telomerase. The above description of necessity provides a limited and merely illustrative sampling of specific compounds, and should not be construed as limiting the scope of the invention. Other features and advantages of the invention will be apparent from the following examples and claims.